Method for establishing a transgenic system of stevia inbred line seeds

Abstract

The present invention discloses a method for establishing a transgenic system for stevia inbred seeds. The method comprises: taking germinating cotyledons of stevia inbred seeds as explants; placing the cotyledons in an Agrobacterium infection solution for infection culture; co-culturing the infected cotyledons and then performing screening culture; performing adventitious bud screening culture and rooting culture on light yellow, plump, and significantly swollen resistant callus; and finally, determining whether the stevia regenerated seedlings are transgenic-positive plants by GUS staining and PCR, thereby obtaining transgenic stevia plants. The present invention uses stevia inbred seeds as test materials and combines a specific callus induction medium, a co-cultivation medium, a resistant callus screening medium, and an adventitious bud screening medium to construct an Agrobacterium-mediated stevia transgenic system. This method not only can obtain transgenic stevia plants, but also greatly shortens the transformation time and improves the transformation rate.

Description

Technical Field

[0001] The present invention relates to the technical field of plant transgenic technology, and in particular to a method for establishing a stevia inbred line seed transgenic system. Background Art

[0002] Stevia ( Stevia rebaudiana Stevia rebaudiana, also known as Stevia rebaudiana, is a perennial herbaceous plant in the Asteraceae family, native to South America. Stevia rebaudiana has attracted considerable attention for its high sweetness (approximately 150-450 times sweeter than sucrose) and low calorie content (only 1 / 300 of sucrose) in its leaves. Steviosides, commonly known as steviosides, are tetracyclic diterpenoid glycosides based on steviol as the aglycone. These include over 30 structurally similar components with distinct properties, including stevioside, steviolbioside, rubusoside, dulcoside A, rubaudioside A (RA), rubaudioside D (RD), and rubaudioside M (RM). Stevioside, as a natural non-nutritive sweetener, has been used in food, beverages, medicine and other fields. In addition, more and more studies have found that stevioside has medicinal value in preventing and assisting in the treatment of obesity, diabetes, hypertension, hyperglycemia, hyperlipidemia, as well as anti-inflammatory, antibacterial, antioxidant and immunity-enhancing effects.

[0003] Since the first introduction of stevia in 1976, my country has continuously engaged in stevia breeding, cultivation, and stevioside processing. Currently, my country leads the world in stevia cultivation area, stevioside production, and export volume, with over 80% of the world's stevioside produced in my country. With the improvement of living standards and the pursuit of a healthier diet, stevioside has attracted increasing attention as an emerging health-conscious sweetener, particularly for special populations such as diabetics, making it a promising application prospect in the food industry. However, severe production shortages hinder its wider and more widespread application. Therefore, increasing stevia leaf yield and the content of steviol glycosides in leaves are urgent challenges in the development and utilization of stevia. The establishment of a stevia transgenic technology system is essential for stevia improvement and can significantly shorten the breeding process. Although stevia tissue culture and rapid propagation techniques are mature, and stevia transgenic systems have been reported, most of them use callus induction from leaves or stem segments before infection, resulting in long production cycles and low plant regeneration rates. Transgenic technology and its receptor systems still need improvement. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for establishing a transgenic system of stevia inbred seeds. Using stevia inbred seeds as materials can overcome the constraints of explant callus induction, the material collection is not limited by time and quantity, and the transformation efficiency is high.

[0005] In order to achieve the above-mentioned object of the invention, the specific technical solutions adopted by the present invention are as follows:

[0006] A method for establishing a transgenic system of stevia inbred line seeds, characterized by comprising the following steps:

[0007] (1) After cleaning and disinfecting, stevia inbred seeds were placed on a culture dish containing moistened sterile filter paper for germination to obtain inbred seedlings;

[0008] (2) Cut off the cotyledons of the seedlings and place them in the Agrobacterium infection solution for vacuum infection;

[0009] (3) After the infection is complete, the infection solution is discarded, the water is dried with sterile filter paper, and the infected cotyledons are placed in the co-cultivation medium for dark culture to obtain expanded cotyledon calli;

[0010] The co-culture medium is: 2.0 mg / L 6-BA + 2.0 mg / L NAA + 20 mg / L As + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8;

[0011] (4) After the co-cultivation is completed, the cotyledon callus is washed and dried, and then the cotyledon callus is transferred to the resistance callus screening medium for screening and culture until the callus tissue has obvious proliferation;

[0012] The resistant callus screening medium is: 1.0 mg / L 6-BA + 0.5 mg / L NAA + 8 mg / L Hyg + 200 mg / L Timentin + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8;

[0013] (5) transferring the light yellow, plump, and obviously swollen resistant callus to a resistant adventitious bud screening medium for adventitious bud screening and culture to obtain adventitious buds;

[0014] The adventitious bud screening medium is 2.0 mg / L 6-BA + 0.2 mg / L IAA + 8 mg / L Hyg + 200 mg / L Timentin + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8;

[0015] (6) cutting the adventitious buds and transferring them to a rooting medium for rooting culture. When the young roots are 2 to 3 cm long, hardening and transplanting are performed to obtain transgenic stevia seedlings.

[0016] The rooting medium is 0.1 mg / L IBA + 8 mg / L Hyg + 200 mg / L Timentin + 1 / 2MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8;

[0017] (7) Identify whether the transgenic stevia seedling is a positive plant and obtain a transgenic stevia plant.

[0018] The present invention can use the GUS staining method to determine the transgenic positive plants.

[0019] Furthermore, in step (1), the stevia inbred line seeds are independently cultivated 'Sweet Seed No. 2' seeds, which are inbred line seeds that have been forced to self-pollinate and passed down through more than 6 generations of single seed, and the plant phenotype and quality properties such as glycoside content are highly consistent.

[0020] Furthermore, in step (2), the OD of the Agrobacterium culture solution is 600 If the concentration of Agrobacterium is low, the amount of bacteria infecting the explants is small, which will lead to a low transformation rate; if the concentration of Agrobacterium is high, the excessive reproduction of Agrobacterium will easily damage the material, which will also reduce the transformation rate.

[0021] Furthermore, in step (2), the vacuum infection is carried out in a sterile centrifuge tube, and the manual vacuuming method is to insert a sterile syringe into the air layer of the centrifuge tube containing the infection liquid, and manually vacuum until the cotyledons are completely water-soaked.

[0022] Furthermore, in step (3), during the co-cultivation, a sterile filter paper is placed between the culture medium and the infected cotyledons to prevent excessive growth of Agrobacterium; the co-cultivation conditions are: culturing in the dark at 20-25°C for 3 days.

[0023] Furthermore, in step (4), after the co-cultivation is completed, the cotyledon callus is washed with sterile water containing 200 mg / L Timentin for 5 times, and the last time is soaked for 10 minutes; the conditions of the screening culture are: 25-28°C, photoperiod 12 h / d, and illumination 1500-2000 Lux. During the screening culture, the callus is subcultured once every two weeks until there is obvious proliferation of callus tissue. Generally, some calli will show obvious proliferation after about 15-20 days.

[0024] Furthermore, in step (5), the conditions for the adventitious bud screening and culture are: 25-28°C, photoperiod 12 h / d, illumination 1500-2000 Lux, and subculture every two weeks.

[0025] Furthermore, in step (6), when the adventitious buds grow to 0.5-1 cm, the adventitious buds are cut off and inserted into a rooting culture medium; the rooting culture conditions are: 25-28°C, a photoperiod of 12 h / d, and an illumination of 1500-2000 Lux.

[0026] Furthermore, in step (6), when the roots are 2 to 3 cm long, the seedlings are removed from the culture medium, the culture medium attached to the surface of the roots is washed off, and the seedlings are transplanted into a seedling tray with a plant spacing of 1.5 to 2.0 cm. The transplanting medium is peat: vermiculite: perlite = 1:1:1. Water is sprayed with a watering can, and the seedlings are covered with plastic wrap to keep them moist. The seedlings are placed in an artificial climate box for cultivation with a set temperature of 25 to 28°C, a photoperiod of 12 h / d, a humidity of 60%, and an illumination of 1500 to 2000 Lux. The plastic wrap is removed after one week, and the seedlings are transplanted with soil after they survive.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses stevia inbred line seeds in combination with specific callus induction medium, co-cultivation medium, resistance callus screening medium and adventitious bud screening medium to construct an Agrobacterium-mediated stevia transgenic system, which can not only obtain stevia transgenic plants but also greatly shorten the transformation system time and improve the transformation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the process for establishing the Stevia inbred line seed transgenic system in Example 1. DETAILED DESCRIPTION

[0030] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0031] Example 1

[0032] A method for establishing a transgenic system of stevia inbred line seeds, comprising the following specific steps:

[0033] (1) After cleaning and disinfecting, the seeds of the Stevia inbred line 'Tianzhong No. 2' were placed in a culture dish containing moistened sterile filter paper and germinated for 4 days to obtain inbred line seedlings with expanded cotyledons;

[0034] (2) Take out the seedlings and cut off the cotyledons. Place the cotyledons in a centrifuge tube containing Agrobacterium infection solution and use a syringe to manually vacuum infect. The Agrobacterium infection solution contains OD 600About 0.4 of Agrobacterium GV3101 was prepared using the plant expression vector pCAMBIA1305, which contains hygromycin (Hyg) resistance and a GUS tag. The Agrobacterium infection solution was prepared using conventional methods in the art.

[0035] (3) After infection, the infection solution was discarded, the water was dried with sterile filter paper, and the infected cotyledons were placed in the co-cultivation medium for co-cultivation;

[0036] The co-cultivation medium was 2.0 mg / L 6-BA + 2.0 mg / L NAA + 20 mg / L As + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8. A sterile filter paper was placed on the medium to prevent excessive growth of Agrobacterium. Each culture dish could hold 20–30 infected cotyledons. The co-cultivation conditions were: incubation at 20–25°C in the dark for 3 days.

[0037] (4) After the co-cultivation, the cotyledon callus was washed with sterile water containing 200 mg / L Timentin for 5 times, with the last time soaked for 10 min. After washing, the water on the cotyledon callus was washed dry with filter paper and then transferred to the resistant callus screening medium for screening culture until the callus tissue showed obvious proliferation and the callus proliferation rate was 60%;

[0038] The resistant callus screening medium is: 1.0 mg / L 6-BA + 0.5 mg / L NAA + 8 mg / L Hyg + 200 mg / L Timentin + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8. A sterile filter paper should be placed on the medium in advance. The screening culture conditions are: 25-28°C, 12 h / d photoperiod, and 1500-2000 Lux. Subculture is performed every two weeks during the screening culture period. Generally, some calli will show significant proliferation after 15-20 days.

[0039] (5) Transferring the light yellow, plump, and obviously swollen resistant callus to a resistant adventitious bud screening medium for adventitious bud screening and culture to obtain callus containing adventitious buds; the adventitious bud induction rate is about 50%;

[0040] The adventitious bud screening culture medium is 2.0 mg / L 6-BA + 0.2 mg / L IAA + 8 mg / L Hyg + 200mg / LTimentin + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8; the conditions for adventitious bud screening culture are: 25-28℃, photoperiod 12 h / d, illumination 1500-2000 Lux, subculture once every two weeks, generally green buds will grow on the callus in about 15-20 days, and adventitious buds will slowly grow out. If the callus grows very large, it can be broken open with tweezers for continued culture, and the brown dead part should be peeled off in time; after about 30 days of light culture, the induction rate of adventitious buds about 0.5 cm in length can reach 50%.

[0041] (6) When the adventitious buds grow to 0.5-1 cm, they are cut off and transferred to rooting medium for rooting culture, with a rooting rate of 99%. When the young roots are 2-3 cm long, they are hardened and transplanted to obtain regenerated seedlings with a survival rate of 90%.

[0042] The rooting medium was 0.1 mg / L IBA + 8 mg / L Hyg + 200 mg / L Timentin + 1 / 2 MS +30 g / L sucrose + 6 g / L agar powder, pH 5.8; the rooting culture conditions were the same as those for callus induction; the seedling hardening and transplanting matrix was peat: vermiculite: perlite = 1:1:1, and water was sprayed thoroughly with a watering can; the seedling hardening and transplanting culture conditions were: cultured in an artificial climate chamber with a set temperature of 25-28℃, a photoperiod of 12 h / d, a humidity of 60%, and an illumination of 1500-2000 Lux. Cover with plastic wrap to keep it moist, remove the plastic wrap after one week, and transplant with soil after survival.

[0043] (7) Determine whether the stevia regenerated seedlings are transgenic positive plants by GUS staining and PCR methods, and obtain stevia transgenic plants.

[0044] The experimental results show that the cycle of the entire transgenic system is 2 to 4 months, which greatly shortens the time of the transformation system.

[0045] Obviously, the above embodiments are merely examples for illustrative purposes and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. However, any obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for establishing a transgenic system of Stevia inbred seeds, characterized in that: The following steps are involved: (1) After cleaning and disinfecting, stevia inbred seeds were placed in a culture dish containing moistened sterile filter paper for germination to obtain inbred seedlings; (2) Cut off the cotyledons of the seedlings and place them in a centrifuge tube containing Agrobacterium infection solution for vacuum infection; The OD600 value of the Agrobacterium infection solution is 0.4; (3) After the infection is complete, the infection solution is discarded, the water is dried with sterile filter paper, and the infected cotyledons are placed in the co-cultivation medium for dark culture to obtain expanded cotyledon calli; The co-culture medium is: 2.0 mg / L 6-BA + 2.0 mg / L NAA + 20 mg / L As + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8; (4) After the co-cultivation is completed, the cotyledon callus is washed and dried, and then the cotyledon callus is transferred to the resistance callus screening medium for screening and culture until the callus tissue has obvious proliferation; The resistant callus screening medium is: 1.0 mg / L 6-BA + 0.5 mg / L NAA + 8 mg / L Hyg + 200 mg / L Timentin + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8; (5) transferring the light yellow, plump, and obviously swollen resistant callus to a resistant adventitious bud screening medium for adventitious bud screening and culture to obtain adventitious buds; The adventitious bud screening medium is 2.0 mg / L 6-BA + 0.2 mg / L IAA + 8 mg / L Hyg + 200 mg / L Timentin + MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8; (6) cutting the adventitious buds and transferring them to a rooting medium for rooting culture. When the young roots are 2 to 3 cm long, hardening and transplanting are performed to obtain transgenic stevia seedlings. The rooting medium is 0.1 mg / L IBA + 8 mg / L Hyg + 200 mg / L Timentin + 1 / 2 MS + 30 g / L sucrose + 6 g / L agar powder, pH 5.8; (7) identifying whether the transgenic stevia seedling is a positive plant, and obtaining a transgenic stevia plant; In step (3), during the co-cultivation, a sterile filter paper is placed between the culture medium and the infected cotyledons to prevent excessive growth of Agrobacterium; the co-cultivation conditions are: culturing in the dark at 20-25°C for 3 days; In step (4), after the co-cultivation is completed, the cotyledon callus is washed with sterile water containing 200 mg / L Timentin, and washed 5 times, with the last time being soaked for 10 minutes; the conditions of the screening culture are: 25-28°C, a photoperiod of 12 h / d, and an illumination of 1500-2000 Lux. During the screening culture period, the callus is subcultured once every two weeks until there is obvious proliferation of callus tissue; In step (5), the conditions for the adventitious bud screening and culture are: 25-28°C, a photoperiod of 12 h / d, an illumination of 1500-2000 Lux, and subculture every two weeks; In step (6), when the adventitious buds grow to 0.5-1 cm, the adventitious buds are cut off and inserted into a rooting culture medium; the rooting culture conditions are: 25-28°C, a photoperiod of 12 h / d, and an illumination of 1500-2000 Lux; In step (6), when the roots are 2 to 3 cm long, the seedlings are removed from the culture medium, the culture medium attached to the root surface is washed off, and the seedlings are transplanted into a seedling tray with a plant spacing of 1.5 to 2.0 cm. The transplanting medium is peat: vermiculite: perlite = 1:1:

1. Water is sprayed with a watering can, and the seedlings are covered with plastic wrap to keep them moist. The seedlings are placed in an artificial climate box for cultivation with a set temperature of 25 to 28°C, a photoperiod of 12 h / d, a humidity of 60%, and an illumination of 1500 to 2000 Lux. The plastic wrap is removed after one week, and the seedlings are transplanted with soil after they survive.

2. The method for establishing a transgenic system of Stevia inbred seeds according to claim 1, wherein: In step (2), the vacuum infection method is to insert a sterile syringe into the air layer of the centrifuge tube containing the infection solution, and manually vacuum the tube until the cotyledons are completely water-soaked.

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